The nociceptive response induced by different classes of Tityus serrulatus neurotoxins: The important role of Ts5 in venom-induced nociception

Scorpion sting envenomations (SSE) are feared by the intense pain that they produce in victims. Pain from SSE is triggered mainly by the presence of neurotoxins in the scorpion venom that modulates voltage-gated ion channels. In Brazil, SSE is mostly caused by Tityus serrulatus, popularly known as yellow scorpion. Here, we evaluated experimental spontaneous nociception induced by T. serrulatus venom as well as its isolated neurotoxins Ts1, Ts5, Ts6, Ts8, and Ts19 frag II, evidencing different degrees of pain behavior in mice. In addition, we developed a mice-derived polyclonal antibody targeting Ts5 able to neutralize the effect of this neurotoxin, showing that Ts5 presents epitopes capable of activating the immune response, which decreased considerably the nociception produced by the whole venom. This is the pioneer study to explore nociception using different classes of T. serrulatus neurotoxins on nociception (α-NaTx, β-NaTx, α-KTx, and β-KTx), targeting potassium and sodium voltage-gated channels, besides demonstrating that Ts5 plays an important role in the scorpion sting induced-pain.


Introduction
In Brazil, scorpion sting envenomations (SSE) are considered as a public health problem reaching more than 105,000 occurrences in 2021 [1]. Tityus serrulatus (Ts), popularly known as yellow scorpion, is the species responsible for most Brazilian SSE cases, mainly due to its adaptation to urban environments and its parthenogenic reproduction [2][3][4]. Moreover, it is considered the most dangerous scorpion in the country, causing considerable morbidity and lethality [3].
The signals and symptoms following Ts stings may be mild, moderate, or severe. During mild envenomings, victims report intense pain, which may come with paresthesia, nausea, and sweating. Moderate envenomings are represented by mild signs and symptoms along with bradycardia or tachycardia, hypo, or hypertension, vomiting, diarrhea, and dyspnea. The severe envenomings are characterized by cardiac failure, pulmonary edema, and death, mainly affecting children and elderly [2,5].
Here, we evaluated experimental spontaneous nociception induced by TsV as well as by its isolated neurotoxins Ts1, Ts5, Ts6, Ts8, and Ts19 frag II. In addition, we developed a micederived polyclonal antibody targeting Ts5 in the attempt to abolish the nociception produced by this toxin in the whole venom. The approach used here besides helping to understand the mechanisms of pain in the SSE and opens the way to potential treatments to minimize the harmful effects for Ts-envenomed patients.

Ethics statement
All protocols were approved by the Institutional Committee for Animal Care and Use of the University of São Paulo (n. 97/2015/USP) and were performed following ethical guidelines established for investigations of experimental pain in conscious animals.

In vivo nociceptive assay
Animals. Male C57BL mice (22-26 g) were obtained from the housing facilities of the Bioterium of Ribeirão Preto Campus of University of São Paulo (USP). Animals were maintained in a temperature-controlled room (22 ± 1˚C) under a 12 h light/12 h dark cycle with free access to food and water. All protocols were performed following ethical guidelines established for investigations of experimental pain in conscious animals [34]. Mass, concentrations, and molarity of tested toxins are summarized in Table 2.

Spontaneous nociception
For spontaneous nociceptive test, animals (n = 6) were challenged with intraplantar injection of TsV or its isolated toxins (Ts1, Ts5, Ts6, Ts8, and Ts19 frag II) using 0.5 μg/paw (right hind paw). Ts5 nociceptive response was also tested using different concentrations of the toxin (0.1, 0.25, 0.5 and 4 μg/paw). Control animals were challenged by the injection of saline solution (vehicle). All animals were observed for 35 min to record the time in which the animals spent either licking or lifting/shaking the injected paw. The spontaneous nociception assay was repeated with TsV after neutralizing Ts5 from the venom (see method of Ts5 neutralization below).

Generation of polyclonal antibodies targeting Ts5
Mice immunization. For mice immunization, 3 Balb/c male mice (18-22 g) were challenged based to previous protocol [35], with modifications. Briefly, each animal received intramuscularly (i.m.) 7.5 μg of Ts5 antigen emulsified in a solution containing complete Freunds' adjuvant, followed by two sequential i.m. boosts with 3-week intervals of 7.5 μg and 5 μg/animal, respectively. The second boost with Ts5 was diluted in a solution with incomplete Freunds' adjuvant, and the third consist only by the antigen (Ts5). Control animals (n = 2) received solely saline solution (vehicle).

ELISA
For monitoring immunization, a standard indirect ELISA was performed. A 96-well plate (ref. 3590, Corning, New York, EUA) was coated with Ts5 (2 μg/well, n = 2) in 0.05 M carbonate/ bicarbonate buffer, pH 9.6 (100 μL/well), and incubated overnight at 4˚C. As positive control,  After verifying the anti-Ts5 antibody development and perform the whole IgGs isolation from the immunized mice serum, the same ELISA described above assay was reproduced with purified IgGs.

Ts5 neutralization
To neutralize the Ts5 from the whole TsV, we considered the percentage of Ts5 recovery from TsV (~2%) [27]. Based on that, we calculate anti-Ts5 IgGs 2-times the equimolar (eqM) quantity of Ts5 from the whole venom. A total of 0.5 μg/animal paw of TsV were used in positive control animals. In the tested group of animals, 0.5 μg of Ts5/animal paw (33.5 pmoles and 241 ng) was pre-incubated 1 h at 37˚C with 2 eqM of IgGs anti-Ts5 (67 pmoles and 10.05 μg). Knowing that IgGs are bivalents, hypothetically, the ratio of binding site and antigen would be 4:1 (two Ts5 binding sites for each IgG). The same rate was used in the TsV and anti-Ts5 group, using 0.5 μg of TsV/animal paw pre-incubated 1 h at 37˚C with IgGs anti-Ts5 (2 eqM of Ts5).

Immunorecognition of Ts5 toxin by heterologous scorpion antivenom and B-cell epitope prediction
A new indirect ELISA was performed as described in 2.3.3 item to evaluate the ability of the commercial scorpion antivenom to recognize Ts5. Plates were coated with TsV and Ts5 (2 μg/ well, n = 2) in 0.05 M carbonate/bicarbonate buffer, pH 9.6 (100 μL/well), and incubated overnight at 4˚C. Scorpion antivenom from Butantan Institute was used as primary antibody (diluted 1:100 in 1% MPBS). As positive control, a non-immune horse serum was used to coat the plate, while as negative controls, wells were coated with TsV and Ts5, and non-immune horse serum was used as primary antibody (diluted 1:100 in 1% MPBS). As secondary antibody, rabbit anti-horse-HRP (A6917, Sigma-Aldrich, St. Louis, MO, USA) antibodies were used. B-cell epitope prediction was performed with ABCpred Server tool (http://crdd.osdd.net/ raghava/abcpred/) [37], with a window length of 14 residues, a threshold of 0.7, and the Ts5 amino acid sequence already deposited on database (Uniprot ID: P46115) [32].

T. serrulatus venom and toxins induce spontaneous nociception
The nociceptive response after TsV and toxins injections showed different degrees of pain behavior in mice ( Fig 1A). As expected, toxins acting on Nav channels (Ts1 and Ts5) induced significant nociceptive behavior after 10 min of injection ( Fig 1B). Ts8, a Kv toxin that modulated Kv4.2 channel, also induced a significant and delayed nociceptive behavior, with the peak of pain behavior observed after 25 min of injection. The other toxins tested (Ts6 and Ts19 frag II), both toxins binding Kv channels, did not showed significant nociceptive behavior.
Notably, TsV and Ts5 presented a significant and lasting (from 5 to 35 min) nociceptive behavior, with a peak of Ts5 at 10 min and 5 min for TsV and with a similar kinetics. Using different quantities of Ts5, the nociceptive behavior demonstrates to be dose-dependent ( Fig  1C). Based on the comparable nociceptive kinetic profile of TsV and Ts5, we hypothesized that Ts5 would account for the nociceptive response caused by the whole venom. To test this hypothesis, next we generated anti-Ts5 polyclonal neutralizing antibodies.

Generation of anti-Ts5 polyclonal antibodies
From 3 immunized animals (Fig 2A), only one of them (named M2) was able to produce detectable titres of anti-Ts5 antibodies (Fig 2B). The i.m. injection of Ts5 showed to be the best route to the neurotoxin inoculation, since we did not have any anti-Ts5 antibody detection when we used intraperitoneal route. The serum of the M2 mouse was used to isolate specific anti-Ts5 IgGs. Mouse IgGs were purified properly, as can be observed in the SDS-PAGE gel ( Fig 3A). After purification, the IgGs' showed to keep the Ts5 recognition, although in a lower title (Fig 3B).

Nociception response induced by Ts5-neutralized venom
Through the nociception assay after incubation of TsV with the producing anti-Ts5, a significant decrease in the mice nociceptive response was observed, when compared to the animals that received the challenge without pre-incubation with the anti-Ts5 antibody (Fig 4A). Nevertheless, the pain behavior was still significant in this group in comparison to the control, showing that other toxins that compose the venom cocktail also contribute to the nociceptive effect ( Fig 4B).
Regarding to control group that received isolated Ts5 toxin pre-incubated with the anti-Ts5 antibodies, the nociceptive response was completely abolish, indicating that the antibodies produced by the immunized M2 mouse were able to recognize Ts5 and neutralize its induced nociception.

Ts5: Immunorecognition and prediction of B-cell epitopes
From indirect ELISA assay, it was demonstrated that scorpion antivenom was able to recognize TsV and Ts5 (Fig 5A), indicating that the specific Brazilian scorpion antivenom is also effective for the treatment of pain in envenoming victims. The in silico analysis with Ts5 toxin suggested 4 predicted epitopes (Fig 5B), that could be responsible to antibody binding (Fig 5C).

Discussion
SSE cause diverse and complex clinical manifestations ranging from local effect to systemic inflammatory reaction, similar to those associated with systemic inflammatory response syndrome and acute sepsis [41]. Scorpion venom is composed of a mixture of molecules, including ion channel-modulating toxins (i.e., neurotoxins) [5,16,20]. Neurotoxins can act on sodium, potassium, calcium, and chloride channels being able to induce a high flux of neurotransmitters that is considered the major driver of the pathology following a scorpion sting [6]. Moreover, many of the toxins that affect ion channels are also able to activate the immune system inducing exacerbated inflammatory responses, which is a research area that has been recently explored [13,14,20,42].

Ts5 role in venom-induced nociception
Immediately after a sting caused by Ts scorpion, local pain is the primary manifestation and, although further studies are needed to lighten this event, cutaneous hyperalgesia and local swelling result in a quick local inflammation with the production of pro-inflammatory mediators, amplifying the pain intensity [14]. For instance, Ts5 toxin acts as a pro-inflammatory mediator through its ability to stimulate the release of macrophage TNF-α and IL-6 [16]. Moreover, TsV have showed the ability to affect all biological systems such as nervous, cardiac,  (2 μg) and tested with non-immune mouse serum; C3(+): wells sensitized with mouse non-immune serum; 1:100 and 1:50: wells sensitized with Ts5 (2 μg) and tested with purified mouse anti-Ts5 IgGs. Rat anti-mouse-HRP antibodies were used in the ELISA detection. The ELISA assay was performed in duplicate, and results are plotted as mean ± SD. Data was analyzed using one-way ANOVA and Tukey's post hoc test. Graph was generated using GraphPad Prism Version 9.3.1. � p < 0.05, when compared to negative controls.
https://doi.org/10.1371/journal.pntd.0011057.g003 pulmonary, urinary, digestive, reproductive, muscular, and the immune system [5], which makes TsV a potent lethal cocktail. In this work we performed assays to elucidate how TsV and its different classes of neurotoxins can act on nociceptive response.
Nociception can be defined as the detection of actual or threatened tissue injury; the specialized neural apparatus fulfilling this role is highly evolutionary conserved, providing a key protective role to the organism [43]. Nociceptors are specialized primary sensory neurons innervating target tissue, such as skin and muscle, which detect injurious stimuli (extremes of temperature, high pressure, endogenous mediators, scorpion stings, among others) [44]. Each of these neurons expresses unique repertoires of voltage-gated sodium channels which are key determinants of excitability integrating the propagation of action potentials to the central nervous system and neurotransmitter release [45], and the activation of the nociceptive system will give rise to the perception of pain.
This work pioneering investigated the nociception of different classes of Ts neurotoxins (Ts1: β-NaTx; Ts5: α-NaTx; Ts6: α-KTx; Ts8 and Ts19: β-KTx). The β-NaΤx (such as Ts1), similarly to α-NaTx counterparts, can compete for binding sites on both insect and mammalian Nav channels. Indeed, the α-NaTx Ts5 demonstrated to bind to both mammalian and insect channels [16,46]. In our work, Ts5 was able to promote intense and dose-dependent spontaneous nociception, with a similar profile triggered by TsV, differently from the other tested toxins (Ts1, Ts6, Ts8, and Ts19 frag II). Among the toxins tested, Ts8 is the only one with a role on Kv4.2 being able to induce pain [23]. However, the experimental pain behavior induced by Kv4.2 showed to be mild and prolonged, different from the pain observed after Ts5 administration.
In a former study, authors speculated that TsV could promote a marked ipsilateral nociceptive response, characterized by thermal and mechanical allodynia and paw licking behaviour. Interestingly, that nociception was inhibited by intraperitoneal injection of anti-inflammatory drugs such as indomethacin, dipyrone, and cyproheptadine, or potent analgesic as morphine, which leads the authors to conclude that the nociceptive response could result from the action of multiple mediators including eicosanoids, histamine, and 5-hydroxytryptamin [11]. We do not discard that hypothesis; however, based on our in vivo assays along with electrophysiological studies, Ts5 activates a nociceptive response immediately after Ts5 injection, inducing an intense pain behaviour in animals (see S1 Video), which could not be a result of mediators of inflammation since they will need time to be produced. Actually, after the stimulus with TsV, macrophages up-regulate the expression of specific receptors, which promote the activation of transcription factors targeting the genes that code for inflammatory mediators leading to a time-consuming inflammatory response [47].
Indeed, using two-electrode voltage-clamp analysis, Ts5 showed to directly inhibit the rapid inactivation of voltage-gated sodium channels related to pain such as Nav 1.3, Nav 1.6, and Nav 1.7 [16]. Such Ts5 activity on a variety of ion channels also explains its high toxicity in mice, although it presents a higher intravenous LD 50 higher than the major TsV-derived toxin, named Ts1 (76 ± 9 μg/kg for Ts1 vs. 94 ± 7 μg/kg for Ts5) [48,49]. Thus, we can infer that among the explored neurotoxins from TsV, Ts5 is the one triggering the most painful response and Ts1 the highest toxicity. Rabbit antihorse-HRP antibodies were used for the ELISA detection. The ELISA assay was performed in duplicate and results are plotted as mean ± SD. Data was analyzed using one-way ANOVA and Tukey's post hoc test. Graph was generated using GraphPad Prism Version 9.3.1. � p < 0.05, when compared to the control group. (B) Ts5 primary sequence (P46115) [32] with the B-cell epitopes predicted by ABCpred Server tool highlighted and their scores. (C) Ts5 molecular model from AlphaFold Protein Structure Database [38,39], modified by PyMol tool [40], with front and back view. Colors (blue, red, orange, and magenta) are not related to the alpha-helix and beta-pleated sheets, they represent the four B-cell epitopes predicted in different types of 3D representation: surface (up) and ribbon (down). https://doi.org/10.1371/journal.pntd.0011057.g005 To verify if the TsV painful stimuli was a result mainly derived from Ts5 effect, we produced anti-Ts5 polyclonal antibodies. Only one of three immunized mice was able to produce antibodies specific to Ts5. Also, as the route of administration dictates the antibody production [50], the i.m. route showed to be important for the production of antibodies anti-Ts5. Since Ts5 presents only 4 predicted epitopes (minimal peptide epitopes), the toxin injection through i.m. route would force antigen presentation to T cells by tissue resident dendritic cells, preventing the induction of T cell tolerance or anergy [51]. In fact, the scorpion antivenom used in Brazil is produced in horses immunized intramuscularly, and we demonstrated here that the horse-derived scorpion antivenom can recognize Ts5.
Analysis of the TSV nociceptive behaviour using anti-Ts5 antibodies, showed that the neutralization of Ts5 from the whole venom was able to significantly reduce the nociception in mice. On the other and, even overturning the Ts5 effect from TsV, the painful stimuli are still significant, demonstrating that other toxins are also contributing to the scorpion painful sting. Therefore, the intense pain sensitivity induced by Ts sting is a result of the binding of different toxins on a gamut of receptors and the induced production of inflammatory mediators.

Conclusion
Our study explored the nociception behavior induced by different classes of scorpion neurotoxins (α-NaTx, β-NaTx, α-KTx, and β-KTx) and demonstrated the important role of Ts5 on Ts painful sting. Thus, in addition to providing new insights to understand the intense pain associated with Ts envenoming, this study could also contribute for the development of novel and specific therapies.